Vaccine for shigella

ABSTRACT

Disclosed are immunogenic conjugates and therapeutic compositions that include such immunogenic conjugates. Also disclosed are methods of treating and/or inhibiting an  Shigella sonnei  infection. The disclosed immunogenic conjugates have the general structure:
 
Pr—Sr—O—N═C-Kdo-OS
 
wherein Pr is a carrier protein, Sr is an optional spacer moiety, Kdo is an 3-deoxy-D-manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide or polysaccharide obtained from  S. sonnei . In specific examples, the immunogenic conjugates include the core oligosaccharide obtained from  S. sonnei  having the structure:
 
     
       
         
         
             
             
         
       
         
         
           
             wherein R is between 1 and 10 disaccharide repeat units. 
           
         
       
    
     In specific examples, the disaccharide repeat unit included in the immunogenic conjugate has the structure:
 
α-L-AltNAcA-3-β-FucNAc4N-4-.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/US2009/053897, filed Aug. 14, 2009, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/089,394, filed Aug. 15, 2008. Theprovisional application is incorporated herein in its entirety.

FIELD

Disclosed herein are immunogenic conjugates made from oligosaccharide orpolysaccharide antigens obtained from Shigella sonnei and methods oftreatment using such conjugates.

BACKGROUND

Shigellae are Gram-negative bacteria, pathogens to humans only, that cancause endemic and epidemic dysentery worldwide, especially in thedeveloping countries. It has been estimated that ≈160 million cases ofshigellosis with ≈1 million deaths occurring worldwide annually. Atleast half of the cases and deaths are believed to occur in children <5years old. Control of this disease is hampered by the low infectiousdose of this pathogen (<100 bacteria) and lack of safe drinking waterand food in the developing world. The symptoms usually start with waterydiarrhea that later develops into dysentery, characterized by highfever, blood and mucus in the stool, and cramps.

Lipopolysaccharides (LPSs) of Shigella are both essential virulencefactors and protective antigens of this genus. The outer domain of thistripartite molecule, termed O-specific polysaccharide (O-SP), “shields”the bacteria from serum complement killing, similar to the action ofcapsular polysaccharides. It has been hypothesized that serum antibodiesto the O-SP of shigellae confer immunity to humans against thehomologous bacteria. To test this hypothesis, experimental vaccinescomposed of protein conjugates of the O-SP of Shigella dysenteriae type1, Shigella sonnei, and Shigella flexneri 2a were synthesized andevaluated in young adults. Evaluation of a S. sonnei O-SP/recombinantPseudomonas aeruginosa Exotoxin A (rEPA) conjugate in Israeli soldiersdemonstrated 72% efficacy with vaccine failures occurring in individualswho responded with significantly lower serum antibody levels than thosewho were protected. Evaluation of such conjugates in children showedage-related antibody responses and protection. A significant improvementin the immunogenicity of S. dysenteriae type 1 conjugates was achievedby using synthetic oligosaccharides (OS) of defined lengths bound bytheir reducing ends to a protein at defined densities. Unfortunately,this improvement could not be replicated for S. sonnei, as synthesis ofS. sonnei O-SP oligosaccharides has not been possible to date. Thus, theneed still exists for improved S. sonnei specific vaccines. Theconjugates and methods of treatment disclosed herein meet those needs.

SUMMARY

Disclosed are immunogenic conjugates and therapeutic compositions thatinclude such immunogenic conjugates. The disclosed immunogenicconjugates have the general structure:Pr—Sr—O—N═C-Kdo-OSwherein Pr is a carrier protein, Sr is an optional spacer moiety, Kdo is3-deoxy-D-manno-octulosonic acid or a derivative thereof, and OS is anoligosaccharide or polysaccharide obtained from S. sonnei. The disclosedimmunogenic conjugate can include a core oligosaccharide obtained fromS. sonnei. In specific examples, the core oligosaccharide obtained fromS. sonnei has the structure:

wherein R is between 1 and 10 disaccharide repeat units.

In some examples, the disaccharide repeat unit included in theimmunogenic conjugate has the structure:α-L-AltNAcA-3-β-FucNAc4N-4-.

Also disclosed are methods of treating and/or inhibiting an infection byS. sonnei in a subject. These methods include by administering to asubject the disclosed immunogenic conjugates, for example by elicitingan immune response in a subject to S. sonnei

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a trace of an exemplary refraction spectra obtained from theelution from a BIOGEL® P-10 gel filtration column of S. sonnei LPS after1% acetic acid hydrolysis. F1-F4 refer to the elution fractions. F1,O-SP, ≈1 RU (Repeat Units); F2, core+average 3.5 RU of O-SP; F3,core+average 1.3 RU of O-SP; F4, degradation products, no core or O-SP.

FIG. 2 is a one dimensional proton nuclear magnetic resonance (NMR)spectrum of S. sonnei O-SP. Integration of the ¹H NMR spectra of S.sonnei O-SP (Top), O-SPC-F2 (Middle), and O-SPC-F3 (Bottom) for thedetermination of the number of RU attached to the core oligosaccharideof S. sonnei. Signals at 5.82 and 5.62 ppm belonging to core α-GalM andα-GalL (1 proton) were integrated with signals at 1.34-1.36 ppmbelonging to FucNAc4N methyl group (3 protons) of O-SP (see Table 3).

FIGS. 3A and 3B are electrospray ionization (ESI) mass spectra of S.sonnei O-SPC-F3 (FIG. 3A) and dephosphorylated O-SPC-F2 (FIG. 3B).Triple charged ions are shown. Components of fraction 2 (F2) with massesof 2514.3 and 2918.1 Daltons (Da) contain one phosphate because ofincomplete dephosphorylation. Assignments of molecular masses are ingiven below in the Examples section.

FIG. 4 is a LPS structure of S. Sonnei referred to herein as scheme 1.

FIG. 5 is the table referred to herein as Table 1.

FIG. 6 is the table referred to herein as Table 2.

FIG. 7 is the table referred to herein as Table 3.

FIG. 8 is the table referred to herein as Table 4. Composition and GM ofserum IgG anti-S. sonnei LPS (Elisa Units, EU) induced by conjugates ofO-SPC bound to bovine serum albumin (BSA), a recombinant diphtheriatoxin (non toxic) (rDT) or a recombinant C. difficile toxin B repeat(rBRU). Mice (10 per group) were injected with 2.5 μg of saccharide as aconjugate per mouse, 3 times, 2 weeks apart and bled one week after thelast two injections.

DETAILED DESCRIPTION I. Abbreviations

-   -   ADH: adipic acid dihydrazide    -   AT: anthrax toxin    -   ATR: anthrax toxin receptor    -   EDAC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl    -   EF: edema factor    -   GLC-MS: gas-liquid chromatography-mass spectrometry    -   kDa: kilodaltons    -   LC-MS: liquid chromatography-mass spectrometry    -   LeTx: lethal toxin    -   LF: lethal factor    -   LOS: lipooligosaccharide    -   LPS: lipopolysaccharide    -   MALDI-TOF: matrix-assisted laser desorption ionization        time-of-flight    -   OS: oligosaccharide    -   μg: microgram    -   μl: microliter    -   PA: protective antigen    -   PBS: phosphate buffered saline    -   SBAP: succinimidyl 3-(bromoacetamido)propionate    -   SFB: succinimidylformylbenzoate    -   SPDP: N-hydroxysuccinimide ester of 3-(2-pyridyl        dithio)-propionic acid    -   SLV: succinimidyllevulinate    -   TT: tetanus toxoid        The saccharide units disclosed herein are abbreviated as below        following conventional oligosaccharide/polysaccharide        nomenclature:    -   AltNAcA: N-Acetyl-L-altrosaminuronic acid    -   anhKdo: anhydro Kdo    -   Fuc: fucose    -   FucNAc4N: 2-acetamido-4-amino-2,4,6-trideoxy-galactose    -   Gal: galactose    -   Glc: glucose,    -   GlcN: glucosamine    -   GlcNAc: N-acetylglucosamine    -   GalNAc: N-acetylgalactosamine    -   Hep: glycero-D-manno-heptopyranoside (heptose)    -   Hex: hexose    -   Man: mannose

II. Listing of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references. Incase of conflict, the present specification, including explanations ofterms, will control.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.It is further to be understood that all nucleotide sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides or other compounds are approximate, andare provided for description. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present disclosure, suitable methods and materials aredescribed below. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

To facilitate review of the various examples of this disclosure, thefollowing explanations of specific terms are provided:

Adjuvant: A substance that enhances the immune response to an antigen.Development of vaccine adjuvants for use in humans is reviewed in Singhet al. (Nat. Biotechnol. 17:1075-1081, 1999), which discloses that, atthe time of its publication, aluminum salts, such as aluminum hydroxide(Amphogel, Wyeth Laboratories, Madison, N.J.), and the MF59microemulsion are the only vaccine adjuvants approved for human use. Analuminum hydrogel (available from Brentg Biosector, Copenhagen, Denmark)is a adjuvant.

In one embodiment, an adjuvant includes a DNA motif that stimulatesimmune activation, for example the innate immune response or theadaptive immune response by T-cells, B-cells, monocytes, dendriticcells, and natural killer cells. Specific, non-limiting examples of aDNA motif that stimulates immune activation include CpGoligodeoxynucleotides, as described in U.S. Pat. Nos. 6,194,388;6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and6,429,199.

Administration: The introduction of a composition into a subject by achosen route. For example, if the chosen route is intranasal, thecomposition is administered by introducing the composition into the noseof the subject.

Animal: A living multi-cellular vertebrate or invertebrate organism, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and veterinary subjects, such as non-human primates.Thus, administration to a subject can include administration to a humansubject. Particular examples of veterinary subjects include domesticatedanimals (such as cats and dogs) and laboratory animals (for example,mice, rabbits, rats, gerbils, guinea pigs, and non-human primates)

Antibody: A protein (or protein complex) that includes one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in a mammal, includingcompositions that are injected, absorbed or otherwise introduced into amammal. Antigens can be any type of biologic molecule including, forexample, simple intermediary metabolites, sugars (e.g.,oligosaccharides, such as oligosaccharides obtained from S. sonnei),lipids, and hormones as well as macromolecules such as complexcarbohydrates (e.g., polysaccharides), phospholipids, nucleic acids andproteins. In some examples, antigens include an oligosaccharide derivedfrom S. sonnei.

Carrier: Any clinically acceptable protein to which an antigen such asan oligosaccharide or polysaccharide can be bound. When bound to acarrier, the bound molecule may become more immunogenic. In someexamples, multiple antigens, for example multiple copies of a singleantigen are bound to a carrier protein, for example in a “sun”configuration in which the antigens radiate away from the center carrierprotein. In other examples, multiple copies of an antigen are bound tothe carrier protein in the ‘lattice configuration.” Carriers are chosento increase the immunogenicity of the bound molecule and/or to elicitantibodies against the carrier which are diagnostically, analytically,and/or therapeutically beneficial. Covalent linking of a molecule to acarrier confers enhanced immunogenicity and T-cell dependence (Pozsgayet al., PNAS 96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18,1976; Dintzis et al., PNAS 73:3671-75, 1976). Useful carriers includepolymeric carriers, which can be natural (for example, proteins frombacteria or viruses), semi-synthetic or synthetic materials containingone or more functional groups to which a reactant moiety can beattached.

Examples of bacterial products for use as carriers include bacterialtoxins, such as B. anthracis PA, LF and LT, and other bacterial toxinsand toxoids, such as tetanus toxin/toxoid, diphtheria toxin/toxoid, P.aeruginosa exotoxin/toxoid/, pertussis toxin/toxoid, and Clostridiumperfringens exotoxin/toxoid, recombinant diphtheria toxin (rDT) andClostridium difficile toxin B repeat (rBRU). Viral proteins, such ashepatitis B surface antigen and core antigen can also be used ascarriers.

Covalent Bond: An interatomic bond between two atoms, characterized bythe sharing of one or more pairs of electrons by the atoms. The terms“covalently bound” or “covalently linked” refer to making two separatemolecules into one contiguous molecule. The terms include reference tojoining an antigen (such as an oligosaccharide obtained from S. sonnei)indirectly to a carrier molecule, with an intervening linker molecule.

Epitope: An antigenic determinant These are particular chemical groupsor contiguous or non-contiguous peptide sequences or saccharide units ona molecule that are antigenic, that is, that elicit a specific immuneresponse. An antibody binds a particular antigenic epitope based on thethree dimensional structure of the antibody and the matching (orcognate) epitope.

Immune Response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus. Animmune response can include any cell of the body involved in a hostdefense response. An immune response includes, but is not limited to, anadaptive immune response or inflammation. In some examples, an immuneresponse is stimulated by administering to a subject a vaccine and/ordisclosed immunogenic conjugate.

Immunogenic Conjugate or Composition: A term used herein to mean acomposition useful for stimulating or eliciting a specific immuneresponse (or immunogenic response) in a vertebrate. In some embodiments,the immunogenic response is protective or provides protective immunity,in that it enables the vertebrate animal to better resist infection ordisease progression from the organism against which the immunogeniccomposition is directed, for example S. sonnei. One specific example ofa type of immunogenic composition is a vaccine.

Immunogen: A compound, composition, or substance which is capable, underappropriate conditions, of stimulating the production of antibodies inan animal, including compositions that are injected or absorbed into ananimal. In some examples, an immunogen is an oligosaccharide obtainedfrom S. sonnei.

Immunologically Effective Dose: An immunologically effective dose of theoligosaccharide-protein or polysaccharide-protein conjugates of thedisclosure is therapeutically effective and will prevent, treat, lessen,or attenuate the severity, extent or duration of a disease or condition,for example, infection by S. sonnei.

Inhibiting or Treating a Disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as S. sonnei infection. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition after it has begun to develop. The term“ameliorating,” with reference to a disease, pathological condition orsymptom, refers to any observable beneficial effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms of the disease in a susceptible subject, areduction in severity of some or all clinical symptoms of the disease, aslower progression of the disease, a reduction in the number of relapsesof the disease, an improvement in the overall health or well-being ofthe subject, or by other parameters well known in the art that arespecific to the particular disease.

Isolated: An “isolated” biological component (such as alipopolysaccharide) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, such as other chromosomal andextra-chromosomal DNA and RNA, proteins, glycolipids and organelles.Isolated does not require absolute purity, and can include protein orpeptide molecules that are at least 50% isolated, such as at least 75%,80%, 90%, 95%, 98%, 99%, or even 100% isolated.

Lipopolysaccharide (LPS): LPS is an endotoxin that is a majorsuprastructure of the outer membrane of Gram-negative bacteria whichcontributes greatly to the structural integrity of the bacteria, andprotects them from host immune defenses. LPS typically contains threecomponents: (a) Lipid A (a hydrophobic domain that typically consists ofa glucosamine disaccharide that is substituted with phosphate groups andlong chain fatty acids in ester and amide linkages); (b) a corepolysaccharide or oligosaccharide that can include, for example,heptose, glucose, galactose and N-acetylglucosamine units depending uponthe genera and species of bacteria; and (c) optionally, polysaccharidedistal or side chain(s) (often referred to as the “O antigen” that caninclude, for example, mannose, galactose, D-glucose,N-acetylgalactosamine, N-acetylglucosamine, L-rhamnose, and adideoxyhexose depending upon the genera and species of bacteria). LipidA and the core polysaccharide or oligosaccharide domains are joinedtogether by one or more units of 3-deoxy-D-manno-octulosonic acid(“Kdo”, also known as ketodeoxyoctonate). A lipooligosaccharide (LOS)(also known as a “short chain LPS”) commonly refers to an LPS thatcontains Lipid A plus a core polysaccharide or oligosaccharide. As usedherein, the term LPS can include short chain LPS and LOS.

Oligosaccharide (OS): The term “oligosaccharide” is not necessarilyrestricted to a molecule having a specific number of saccharide units.However, in general, an oligosaccharide is a carbohydrate that containsfrom about 3 to about 20 simple sugars (e.g., monosaccharides) linkedtogether. O-specific oligosaccharide+core (O-SPC) refers to anO-specific oligosaccharide chain attached to a core oligosaccharide orpolysaccharide chain.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the proteins and othercompositions herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions, powder, pill, tablet, or capsule forms,conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpeptide, protein, conjugate, LPS, or other active compound is one thatis isolated in whole or in part from proteins, lipids or othercontaminants. Generally, substantially purified peptides, proteins,conjugates, LPSs or other active compounds for use within the disclosurecomprise more than 60% of all macromolecular species present in apreparation prior to admixture or formulation of the peptide, protein,conjugate, LPS or other active compound with a pharmaceutical carrier,excipient, buffer, absorption enhancing agent, stabilizer, preservative,adjuvant or other co-ingredient in a complete pharmaceutical formulationfor therapeutic administration. More typically, the peptide, protein,conjugate, LPS or other active compound is purified to represent greaterthan 70%, often greater than 90% of all macromolecular species presentin a purified preparation prior to admixture with other formulationingredients. In other cases, the purified preparation may be essentiallyhomogeneous, wherein other macromolecular species are not detectable byconventional techniques.

Therapeutically effective amount: A quantity of a specific substance(for example, this may be the amount of an S. sonnei O-SPC-protein orpolysaccharide-protein conjugate useful in increasing resistance to,preventing, ameliorating, and/or treating infection and disease causedby S. sonnei) sufficient to achieve a desired effect in a subject beingtreated. Ideally, a therapeutically effective amount of an agent is anamount sufficient to increase resistance to, prevent, ameliorate, and/ortreat infection and disease caused by S. sonnei infection in a subjectwithout causing a substantial cytotoxic effect in the subject. Theeffective amount of an agent useful for increasing resistance to,preventing, ameliorating, and/or treating infection and disease causedby S. sonnei infection in a subject will be dependent on the subjectbeing treated, the severity of the affliction, and the manner ofadministration of the therapeutic composition. For example, atherapeutically effective amount of composition can vary from about 0.01mg/kg body weight to about 1 g/kg body weight. When administered to asubject, a dosage will generally be used that will achieve targetconcentrations shown to achieve a desired in vivo effect.

Shigella: A genus of Gram-negative, non-spore forming rod-shapedbacteria closely related to Escherichia coli and Salmonella. Thecausative agent of human shigellosis, Shigella cause disease inprimates, but not in other mammals. Shigella species are classified byfour serogroups:

Serogroup A: S. dysenteriae (12 serotypes)

Serogroup B: S. flexneri (6 serotypes)

Serogroup C: S. boydii (23 serotypes)

Serogroup D: S. sonnei (1 serotype)

Group A-C are physiologically similar; S. sonnei (group D) can bedifferentiated on the basis of biochemical metabolism assays. ThreeShigella groups are the major disease-causing species: S. flexneri isthe most frequently isolated species worldwide and accounts for 60% ofcases in the developing world; S. sonnei causes 77% of cases in thedeveloped world, compared to only 15% of cases in the developing world;and S. dysenteriae is usually the cause of epidemics of dysentery,particularly in confined populations such as refugee camps.

Toxoid: A nontoxic derivative of a bacterial exotoxin produced, forexample, by formaldehyde or other chemical treatment. Toxoids are usefulin the formulation of immunogenic compositions because they retain mostof the antigenic properties of the toxins from which they were derived.

Vaccine: A vaccine is a pharmaceutical composition that elicits aprophylactic or therapeutic immune response in a subject. In some cases,the immune response is a protective response. Typically, a vaccineelicits an antigen-specific immune response to an antigen of a pathogen,for example, a bacterial viral pathogen.

III. Description of Several Embodiments

This disclosure relates to conjugates for use in eliciting an immuneresponse to S. sonnei and methods of using such conjugates for treatingand/or inhibiting S. sonnei infection, for example by eliciting animmune response targeting S. sonnei oligosaccharides. To enhanceantibody responses of subject, such as young children, to S. sonneiO-SP, and based on the (statistically significant) higher antibodylevels induced by the synthetic S. dysenterie type 1 “sun” configurationconjugates than those induced by the full-length bacterial lattice-typeconjugates disclosed herein are sun configuration S. sonnei conjugates.Because the synthesis of S. sonnei O-SP-based oligosaccharides could notbe extended beyond a disaccharide unit prior to this disclosure, it waspreviously not possible to produce such conjugates.

To overcome this problem, low-molecular mass oligosaccharides containingLPS core plus several O-SP repeat units (RU) (referred to herein asO-SPC) from the bacterial LPS of S. sonnei were isolated and used forconjugate preparation. In one example, an oligosaccharide fraction, F2containing the core plus an average of 3.5 repeat units RU of O-SP, wasbound to the protein carriers by oxime formation. Examples of methods ofconjugating oligosaccharides can be found in International PatentApplication No. PCT/US2007/016373, which is incorporated herein byreference in its entirety.

The disclosed conjugates were antigenic and induced significantly higherIgG antibody levels than those induced by conjugates prepared withfull-length O-SP. The antibodies induced by the O-SPC conjugate weredirected to the RU of the O-SP and not to the core region based on thesimilar antibody levels obtained by using S. sonnei or P. shigelloidesLPS for ELISA and the close to a 100% inhibition of O-SPC-inducedantibodies by O-SP of both organisms. The finding that the identity ofthe sugar residue positioned at the nonreducing end of the synthetic S.dysenterie type 1 oligosaccharide conjugates is an important variablefor the immunogenicity of these conjugates and may be one reason for thesuperior immunogenicity of the O-SPC and synthetic oligosaccharideconjugates over O-SP conjugates; the former have more end groups.

A. Conjugate Vaccines

Disclosed herein are immunogenic conjugates that are formed from thecore oligosaccharide obtained from S. sonnei and carrier protein. Theimmunogenic conjugates have the general structure:Pr—Sr—O—N═C-Kdo-OS  (I)in which Pr represents carrier protein, Sr is an optional spacer moiety,Kdo is 3-deoxy-D-manno-octulosonic acid or a derivative thereof, and OSis an oligosaccharide or polysaccharide obtained from S. sonnei LPS. Insome embodiments, the immunogenic conjugate includes a coreoligosaccharide from S. sonnei. Exemplary methods of obtaining a coreoligosaccharide from S. sonnei are given below in the examples, althoughother suitable methods, such as those detailed in this section can beused.

In some embodiments, the oligosaccharide core comprises or consists of:

wherein R is between 1 and 10 disaccharide repeat units, such as about1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about9, or about 10 disaccharide repeat units. In some examples, thedisaccharide repeat unit comprises or consists of the structure:α-L-AltNAcA-3-β-FucNAc4N-4-.  (III)The linkage between the oligosaccharide core (II) and the disacchariderepeat (III) is between the Glc-3 and the FucNAc4N moieties shown abovein bold.

In some embodiments, multiple O-PSCs are conjugated to a single proteincarrier molecule. For example, the number of oligosaccharide chainsbound to a single protein carrier molecule may vary depending upon thespecific O-SPC and the carrier protein, but in general, about 1 to about20, such as about 1, about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, about 10, about 11, about 12, about 13, about 14,about 15, about 16, about 17, about 18, about 19, about 20 or even morethan 20 O-SPC chains can be bound to each protein carrier molecule, forexample about 1 to about 5, about 3 to about 7, about 5 to about 9,about 7 to about 12, about 9 to about 15, about 13 to about 17 or evenabout 15 to about 20 O-SPC chains can be bound to each protein carriermolecule.

Specific, non-limiting examples of water soluble protein carriers thatcan be conjugated to O-SPC obtained from S. sonnei include, but are notlimited to, natural, semi-synthetic or synthetic polypeptides orproteins from bacteria or viruses. In some examples, bacterial productsfor use as carriers include bacterial wall proteins and other products(for example, streptococcal or staphylococcal cell walls), and solubleantigens of bacteria. In another examples, bacterial products for use ascarriers include bacterial toxins. Bacterial toxins include bacterialproducts that mediate toxic effects, inflammatory responses, stress,shock, chronic sequelae, or mortality in a susceptible host. Specific,non-limiting examples of bacterial toxins include, but are not limitedto: B. anthracis PA (for example, as encoded by bases 143779 to 146073of GENBANK® as Accession No. NC 007322 as available Aug. 15, 2008,herein incorporated by reference), including variants that share atleast 90%, at least 95%, or at least 98% amino acid sequence homology toPA, fragments that contain at least one antigenic epitope, and analogsor derivatives capable of eliciting an immune response; B. anthracis LF(for example, as encoded by the complement of bases 149357 to 151786 ofGENBANK® Accession No. NC 007322 as available Aug. 15, 2008, hereinincorporated by reference); bacterial toxins and toxoids, such astetanus toxin/toxoid (for example, as described in U.S. Pat. Nos.5,601,826 and 6,696,065); diphtheria toxin/toxoid (for example, asdescribed in U.S. Pat. Nos. 4,709,017 and 6,696,065); P. aeruginosaexotoxin/toxoid/ (for example, as described in U.S. Pat. Nos. 4,428,931,4,488,991 and 5,602,095); pertussis toxin/toxoid (for example, asdescribed in U.S. Pat. Nos. 4,997,915, 6,399,076 and 6,696,065); C.difficile toxin B (for example, as available of GENBANK® Accession No.CAA43299, AAO83645, AAO83646, CAC19891 as available Aug. 15, 2008,herein incorporated by reference); and C. perfringens exotoxin/toxoid(for example, as described in U.S. Pat. Nos. 5,817,317 and 6,403,094).Viral proteins, such as hepatitis B surface antigen (for example, asdescribed in U.S. Pat. Nos. 5,151,023 and 6,013,264) and core antigen(for example, as described in U.S. Pat. Nos. 4,547,367 and 4,547,368)can also be used as carriers, as well as proteins from higher organismssuch as keyhole limpet hemocyanin, horseshoe crab hemocyanin, edestin,mammalian serum albumins, and mammalian immunoglobulins. In specificexamples, a disclosed immunogenic conjugate includes a recombinantdiphtheria toxin (rDT). In other examples, a disclosed immunogenicconjugate includes a recombinant aeruginosa Exotoxin A (rEPA). In stillother examples, a disclosed immunogenic conjugate includes a recombinantC. difficile toxin B repeat (rBRU).

Methods for conjugating oligosaccharides or polysaccharides having a3-deoxy-D-manno-octulosonic acid moiety located at the terminal reducingend of the oligosaccharides or polysaccharides can be found inInternational Patent Application No. PCT/US2007/016373, which isincorporated herein by reference in its entirety. According to thesemethods, binding the oligosaccharide by Kdo at the reducing end of theoligosaccharide means that all of the conserved oligosaccharidestructure remains intact or unmodified (e.g., none of the saccharideresidues are oxidized). The conjugates disclosed herein preserve theexternal non-reducing end of the oligosaccharide, are recognized byantisera, and induce antibody responses in mice.

The oligosaccharide may be obtained from S. sonnei, as well as to otherenterobacteriacea and other gram-negative bacteria having Kdo moleculebetween Lipid A and oligo/polysaccharide chain of their LPS. Theoligosaccharides or polysaccharides that are conjugated include ageneral structure of:O-chain-core OS-Kdo  (IV)

The Kdo moiety (or derivative thereof) is the moiety that results afteracid hydrolysis treatment of the isolated LOS or LPS. In some examplesthe acid hydrolysis treatment results in the formation of anhydro-Kdo asdescribed in more detail below and it has a structure represented by(anhydro-Kdo could also be referred to as 4, 8(7)-anhydro derivative ofKdo):

The oligosaccharide or polysaccharide typically is derived from LPSpresent in S. sonnei. The LPS initially is isolated from the otherconstituents of the bacteria cell structure. Illustrative LPS-isolationtechniques are described, for example, below in the examples section andmore generally in Westphal et al., Meth. Carbohydr. Chem. 5:83-89, 1965,which is incorporated herein by reference in its entirety. OtherLPS-isolation techniques include enzyme digestion and alcoholprecipitation, chromatography by gel filtration and ion-exchange.

The isolated LPS then is subjected to mild acid hydrolysis to cleave theLipid A from the polysaccharide or oligosaccharide domain such that the3-deoxy-D-manno-octulosonic acid remains linked to the polysaccharide oroligosaccharide domain. Such techniques are described, for example, inAuzanneau, J. Chem. Soc. Perkin Trans. 1:509-516, 1991 and Rybka et al.,J. Microbiol. Methods 64(2):171-184, 2006, both of which areincorporated herein by reference. Illustrative hydrolysis conditionsinclude treating the LPS with acetic acid for 1-3 hours at about 100°C., or hydrolyzing LPS in a mixture of acetic acid and sodium acetate(e.g., treating 50 mg LPS with a mixture of 73.5 ml of 0.2 M acetic acidand 26.5 ml of 0.2 M sodium acetate for 5 hours at 100° C. in 5 mlvolume). In some examples, the acid hydrolysis transforms the Kdostructure in the isolated LPS to an anhydro-Kdo structure.

Conjugation of the oligosaccharide or polysaccharide to the carrierprotein is accomplished via formation of an oxime linkage between acarbonyl functional group present in the Kdo moiety and an aminooxyfunctional group present on the carrier protein. The oxime linkagereaction is a chemoselective ligation since it involves the aqueouscovalent coupling of unprotected, highly functionalized biomoleculesthat contain at least a pair of functional groups that react togetherexclusively, within a biological environment. Oxime linkages can beformed in an aqueous reaction environment, and are stable, from pH 5 topH 7. Other advantageous features of forming oxime linkages include arelatively short reaction time, a good yield, and formation at ambienttemperature. These conditions avoid denaturation of the carrier protein.

The reactive carbonyl functional group present in the Kdo moiety can bean aldehyde or a ketone remaining after acid hydrolysis cleavage of theLipid A from the LPS. The carrier protein is functionalized with anaminooxy group. The synthetic scheme for forming the oxime linkage isshown below:Pr—Sr—O—NH₂+Kdo-O-SPC→Pr—Sr—O—N═C-Kdo-O-SPC  (V)

wherein Pr is a carrier protein, Sr is an optional spacer moiety, Kdo is-deoxy-D-manno-octulosonic or a derivative thereof, and OS is anoligosaccharide or polysaccharide residue from the cleavage of Lipid Afrom LPS. Condensation between the carbonyl and aminooxy groups leads toa stable oxime linkage between the OS and carrier protein. The spacermoiety may have any structure that is present in the linker reagents asdescribed below. Alternatively, the Kdo-O-SPC structure could be reactedinitially with an aminooxy reagent, and the resultingaminooxy-functionalized reactant could be reacted with the protein.

The oxime conjugation reaction is performed at pH 5 to about pH 7 atambient temperature conditions in an aqueous environment. The reactiontime typically ranges from about 8 to about 24 hours. However, less than100% conjugation completion can be achieved in less than 8 hours, andthe 8-24 hour reaction time assumes near 100% conjugation completion.

The carrier protein (or Kdo-O-SPC or derivitaive thereof) can befunctionalized to include at least one reactive aminooxy moiety byvarious techniques as described, for example, in Kielb et al., J. Org.Chem. 70:6987-6990, 2005 and U.S. Patent Application Publication No.2005/0169941, both of which are incorporated herein by reference.Functionalization of the carrier protein can result in the inclusion ofan optional spacer moiety as noted above. In illustrative examples, acarrier protein (or Kdo-O-SPC) may be reacted with a linker reagent toincorporate the spacer moiety and the aminooxy functional moiety. Thelinker reagent typically is a heterobifunctional compound that includesat least one aminooxy group and a second functional group that isreactive with the carrier protein. Suitable linker reagents includeaminooxy-thiol compounds. Illustrative aminooxy-thiol linker reagentsinclude aminoooxy-alkyl-thiols such as (thiolalkyl)hydroxylamines (e.g.,O-(3-thiolpropyl)hydroxylamine) and aminooxy-aryl-thiols. In the case ofaminooxy-thiol linker reagents, the carrier protein may be treated tointroduce thiol-reactive groups. For example, the carrier protein may betreated with a treatment agent that introduces thiol-reactivehaloacetamido or thiol-reactive maleimido moieties onto the carrierprotein. The haloacetamido-containing protein or maleimido-containingprotein is reacted with the aminooxy-thiol reagent to form theaminooxylated carrier protein via the formation of stable thioetherlinkages.

The amount of oligosaccharide or polysaccharide reacted with the amountof protein may vary depending upon the specific LPS from which the O-SPCis derived and the carrier protein. However, the respective amountsshould be sufficient to introduce about 5-20 chains of O-SPC onto theprotein. In certain examples, the mol ratio of carbonyl groups on O-SPC)to aminooxy groups on the protein may range from about 0.3:1 to about1:3, more particularly 1:1 to about 1:2, and more preferably about 1:1.The resulting number of oligosaccharide chains bound to a single proteincarrier molecule may vary depending upon the specific LPS and thecarrier protein, but in general, about 1 to about 20, such as about 1,about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20 or even more than 20 O-SPC chainscan be bound to each protein carrier molecule. The yield based on theamount of protein ranges from about 70 to about 90% in proteinderivatization step and about 70 to about 90% after the conjugation withthe OS.

Specific, non-limiting examples of water soluble protein carriersinclude, but are not limited to, natural, semi-synthetic or syntheticpolypeptides or proteins from bacteria or viruses. In one embodiment,bacterial products for use as carriers include bacterial wall proteinsand other products (for example, streptococcal or staphylococcal cellwalls), and soluble antigens of bacteria. In another embodiment,bacterial products for use as carriers include bacterial toxins.Bacterial toxins include bacterial products that mediate toxic effects,inflammatory responses, stress, shock, chronic sequelae, or mortality ina susceptible host. Specific, non-limiting examples of bacterial toxinsinclude, but are not limited to: B. anthracis PA (for example, asencoded by bases 143779 to 146073 of GENBANK® Accession No. NC 007322,herein incorporated by reference), including variants that share atleast 90%, at least 95%, or at least 98% amino acid sequence homology toPA, fragments that contain at least one antigenic epitope, and analogsor derivatives capable of eliciting an immune response; B. anthracis LF(for example, as encoded by the complement of bases 149357 to 151786 ofGENBANK® Accession No. NC 007322); bacterial toxins and toxoids, such astetanus toxin/toxoid (for example, as described in U.S. Pat. Nos.5,601,826 and 6,696,065); diphtheria toxin/toxoid (for example, asdescribed in U.S. Pat. Nos. 4,709,017 and 6,696,065); C. difficile toxinA or toxin B (for example, as available of GENBANK® Accession No.CAA43299, AAO83645, AAO83646, CAC19891); P. aeruginosa exotoxin/toxoid/(for example, as described in U.S. Pat. Nos. 4,428,931, 4,488,991 and5,602,095); pertussis toxin/toxoid (for example, as described in U.S.Pat. Nos. 4,997,915, 6,399,076 and 6,696,065); and C. perfringensexotoxin/toxoid (for example, as described in U.S. Pat. Nos. 5,817,317and 6,403,094). Viral proteins, such as hepatitis B surface antigen (forexample, as described in U.S. Pat. Nos. 5,151,023 and 6,013,264) andcore antigen (for example, as described in U.S. Pat. Nos. 4,547,367 and4,547,368) can also be used as carriers, as well as proteins from higherorganisms such as keyhole limpet hemocyanin, horseshoe crab hemocyanin,edestin, mammalian serum albumins, and mammalian immunoglobulins.

Following conjugation of the oligosaccharide or polysaccharide to thecarrier protein, the conjugate can be purified by a variety oftechniques well known to one of skill in the art. One goal of thepurification step is to remove the unbound oligosaccharide orpolysaccharide from the conjugation reaction product composition. Onemethod for purification, involving ultrafiltration in the presence ofammonium sulfate, is described in U.S. Pat. No. 6,146,902.Alternatively, the conjugates can be purified away from unreactedoligosaccharide/polysaccharide and carrier by any number of standardtechniques including, for example, size exclusion chromatography,density gradient centrifugation, hydrophobic interaction chromatography,or ammonium sulfate fractionation. See, for example, Anderson et al., J.Immunol. 137:1181-1186, 1986 and Jennings & Lugowski, J. Immunol.127:1011-1018, 1981. The compositions and purity of the conjugates canbe determined by GLC-MS and MALDI-TOF spectrometry.

B. Therapeutic Formulations.

The conjugates disclosed herein may be included in pharmaceuticalcompositions (including therapeutic and prophylactic formulations),typically combined together with one or more pharmaceutically acceptablevehicles and, optionally, other therapeutic ingredients (for example,antibiotics).

Such pharmaceutical compositions can be administered to subjects by avariety of mucosal administration modes, including by oral, rectal,intranasal, intrapulmonary, or transdermal delivery, or by topicaldelivery to other surfaces. Optionally, the conjugate can beadministered by non-mucosal routes, including by intramuscular,subcutaneous, intravenous, intra-atrial, intra-articular,intraperitoneal, or parenteral routes. Alternatively, the conjugate canbe administered ex vivo by direct exposure to cells, tissues or organsoriginating from a subject.

To formulate the pharmaceutical compositions, the conjugate can becombined with various pharmaceutically acceptable additives, as well asa base or vehicle for dispersion of the conjugate. Desired additivesinclude, but are not limited to, pH control agents, such as arginine,sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like.In addition, local anesthetics (for example, benzyl alcohol),isotonizing agents (for example, sodium chloride, mannitol, sorbitol),adsorption inhibitors (for example, TWEEN® 80), solubility enhancingagents (for example, cyclodextrins and derivatives thereof), stabilizers(for example, serum albumin), and reducing agents (for example,glutathione) can be included. Adjuvants, such as aluminum hydroxide (forexample, AMPHOGEL®, Wyeth Laboratories, Madison, N.J.), Freund'sadjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton,Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), among many othersuitable adjuvants well known in the art, can be included in thecompositions. When the composition is a liquid, the tonicity of theformulation, as measured with reference to the tonicity of 0.9% (w/v)physiological saline solution taken as unity, is typically adjusted to avalue at which no substantial, irreversible tissue damage will beinduced at the site of administration. Generally, the tonicity of thesolution is adjusted to a value of about 0.3 to about 3.0, such as about0.5 to about 2.0, or about 0.8 to about 1.7.

The conjugate can be dispersed in a base or vehicle, which can include ahydrophilic compound having a capacity to disperse the conjugate, andany desired additives. The base can be selected from a wide range ofsuitable compounds, including but not limited to, copolymers ofpolycarboxylic acids or salts thereof, carboxylic anhydrides (forexample, maleic anhydride) with other monomers (for example, methyl(meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers,such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives, such as hydroxymethylcellulose,hydroxypropylcellulose and the like, and natural polymers, such aschitosan, collagen, sodium alginate, gelatin, hyaluronic acid, andnontoxic metal salts thereof. Often, a biodegradable polymer is selectedas a base or vehicle, for example, polylactic acid, poly(lacticacid-glycolic acid) copolymer, polyhydroxybutyric acid,poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof.Alternatively or additionally, synthetic fatty acid esters such aspolyglycerin fatty acid esters, sucrose fatty acid esters and the likecan be employed as vehicles. Hydrophilic polymers and other vehicles canbe used alone or in combination, and enhanced structural integrity canbe imparted to the vehicle by partial crystallization, ionic bonding,cross-linking and the like. The vehicle can be provided in a variety offorms, including fluid or viscous solutions, gels, pastes, powders,microspheres and films for direct application to a mucosal surface.

The conjugate can be combined with the base or vehicle according to avariety of methods, and release of the conjugate can be by diffusion,disintegration of the vehicle, or associated formation of waterchannels. In some circumstances, the conjugate is dispersed inmicrocapsules (microspheres) or nanocapsules (nanospheres) prepared froma suitable polymer, for example, isobutyl 2-cyanoacrylate (see, forexample, Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991), anddispersed in a biocompatible dispersing medium, which yields sustaineddelivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate. For solid compositions, conventional nontoxic pharmaceuticallyacceptable vehicles can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like.

Pharmaceutical compositions for administering the conjugate can also beformulated as a solution, microemulsion, or other ordered structuresuitable for high concentration of active ingredients. The vehicle canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), and suitable mixtures thereof.Proper fluidity for solutions can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of a desired particlesize in the case of dispersible formulations, and by the use ofsurfactants. In many cases, it will be desirable to include isotonicagents, for example, sugars, polyalcohols, such as mannitol andsorbitol, or sodium chloride in the composition. Prolonged absorption ofthe conjugate can be brought about by including in the composition anagent which delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the conjugate can be administered in a timerelease formulation, for example in a composition which includes a slowrelease polymer. These compositions can be prepared with vehicles thatwill protect against rapid release, for example a controlled releasevehicle such as a polymer, microencapsulated delivery system orbioadhesive gel. Prolonged delivery in various compositions of thedisclosure can be brought about by including in the composition agentsthat delay absorption, for example, aluminum monostearate hydrogels andgelatin. When controlled release formulations are desired, controlledrelease binders suitable for use in accordance with the disclosureinclude any biocompatible controlled release material which is inert tothe active agent and which is capable of incorporating the conjugateand/or other biologically active agent. Numerous such materials areknown in the art. Useful controlled-release binders are materials thatare metabolized slowly under physiological conditions following theirdelivery (for example, at a mucosal surface, or in the presence ofbodily fluids). Appropriate binders include, but are not limited to,biocompatible polymers and copolymers well known in the art for use insustained release formulations. Such biocompatible compounds arenon-toxic and inert to surrounding tissues, and do not triggersignificant adverse side effects, such as nasal irritation, immuneresponse, inflammation, or the like. They are metabolized into metabolicproducts that are also biocompatible and easily eliminated from thebody.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-co-glycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat. No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the conjugate in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theconjugate and/or other biologically active agent into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the conjugate plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theprevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

C. Methods of Treatment

In accordance with the various treatment methods of the disclosure, theconjugate can be delivered to a subject in a manner consistent withconventional methodologies associated with management of the disorderfor which treatment or prevention is sought. In accordance with thedisclosure herein, a prophylactically or therapeutically effectiveamount of the conjugate and/or other biologically active agent isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent, inhibit, and/or ameliorate a selecteddisease or condition or one or more symptom(s) thereof.

Typical subjects intended for treatment with the compositions andmethods of the present disclosure include humans, as well as non-humanprimates and other animals. To identify subjects for prophylaxis ortreatment according to the methods of the disclosure, accepted screeningmethods are employed to determine risk factors associated with atargeted or suspected disease of condition (for example, coughingdisease) as discussed herein, or to determine the status of an existingdisease or condition in a subject. These screening methods include, forexample, conventional work-ups to determine environmental, familial,occupational, and other such risk factors that may be associated withthe targeted or suspected disease or condition, as well as diagnosticmethods, such as various ELISA and other immunoassay methods, which areavailable and well known in the art to detect and/or characterizedisease-associated markers. These and other routine methods allow theclinician to select patients in need of therapy using the methods andpharmaceutical compositions of the disclosure. In accordance with thesemethods and principles, a conjugate and/or other biologically activeagent can be administered according to the teachings herein as anindependent prophylaxis or treatment program, or as a follow-up, adjunctor coordinate treatment regimen to other treatments, including surgery,vaccination, immunotherapy, hormone treatment, cell, tissue, or organtransplants, and the like.

The conjugates can be used in coordinate vaccination protocols orcombinatorial formulations. In certain embodiments, novel combinatorialimmunogenic compositions and coordinate immunization protocols employseparate immunogens or formulations, each directed toward eliciting ananti-LPS or an anti-LOS immune response. Separate immunogens that elicitthe anti-LPS or anti-LOS immune response can be combined in a polyvalentimmunogenic composition administered to a subject in a singleimmunization step, or they can be administered separately (in monovalentimmunogenic compositions) in a coordinate immunization protocol.

The administration of the conjugate of the disclosure can be for eitherprophylactic or therapeutic purpose. When provided prophylactically, theconjugate is provided in advance of any symptom. The prophylacticadministration of the conjugate serves to prevent or ameliorate anysubsequent infection. When provided therapeutically, the conjugate isprovided at (or shortly after) the onset of a symptom of disease orinfection. The conjugate of the disclosure can thus be provided prior tothe anticipated exposure to S. sonnei so as to attenuate the anticipatedseverity, duration or extent of an infection and/or associated diseasesymptoms, after exposure or suspected exposure to the bacteria, or afterthe actual initiation of an infection.

For prophylactic and therapeutic purposes, the conjugate can beadministered to the subject in a single bolus delivery, via continuousdelivery (for example, continuous transdermal, mucosal or intravenousdelivery) over an extended time period, or in a repeated administrationprotocol (for example, by an hourly, daily or weekly, repeatedadministration protocol). The therapeutically effective dosage of theconjugate can be provided as repeated doses within a prolongedprophylaxis or treatment regimen that will yield clinically significantresults to alleviate one or more symptoms or detectable conditionsassociated with a targeted disease or condition as set forth herein.Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby administration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject.Suitable models in this regard include, for example, murine, rat,porcine, feline, non-human primate, and other accepted animal modelsubjects known in the art. Alternatively, effective dosages can bedetermined using in vitro models (for example, immunologic andhistopathologic assays). Using such models, only ordinary calculationsand adjustments are required to determine an appropriate concentrationand dose to administer a therapeutically effective amount of theconjugate (for example, amounts that are effective to elicit a desiredimmune response or alleviate one or more symptoms of a targeteddisease). In alternative embodiments, an effective amount or effectivedose of the conjugate may simply inhibit or enhance one or more selectedbiological activities correlated with a disease or condition, as setforth herein, for either therapeutic or diagnostic purposes.

The actual dosage of the conjugate will vary according to factors suchas the disease indication and particular status of the subject (forexample, the subject's age, size, fitness, extent of symptoms,susceptibility factors, and the like), time and route of administration,other drugs or treatments being administered concurrently, as well asthe specific pharmacology of the conjugate for eliciting the desiredactivity or biological response in the subject. Dosage regimens can beadjusted to provide an optimum prophylactic or therapeutic response. Asdescribed above in the forgoing listing of terms, a therapeuticallyeffective amount is also one in which any toxic or detrimental sideeffects of the conjugate and/or other biologically active agent isoutweighed in clinical terms by therapeutically beneficial effects. Anon-limiting range for a therapeutically effective amount of a conjugateand/or other biologically active agent within the methods andformulations of the disclosure is about 0.01 mg/kg body weight to about10 mg/kg body weight, such as about 0.05 mg/kg to about 5 mg/kg bodyweight, or about 0.2 mg/kg to about 2 mg/kg body weight.

Upon administration of a conjugate of the disclosure (for example, viainjection, aerosol, oral, topical or other route), the immune system ofthe subject typically responds to the immunogenic composition byproducing antibodies specific for LPS, LOS and/or an antigenic epitopepresented by the conjugate. Such a response signifies that animmunologically effective dose of the conjugate was delivered. Animmunologically effective dosage can be achieved by single or multipleadministrations (including, for example, multiple administrations perday), daily, or weekly administrations. For each particular subject,specific dosage regimens can be evaluated and adjusted over timeaccording to the individual need and professional judgment of the personadministering or supervising the administration of the conjugate. Insome embodiments, the antibody response of a subject administered thecompositions of the disclosure will be determined in the context ofevaluating effective dosages/immunization protocols. In most instancesit will be sufficient to assess the antibody titer in serum or plasmaobtained from the subject. Decisions as to whether to administer boosterinoculations and/or to change the amount of the composition administeredto the individual can be at least partially based on the antibody titerlevel. The antibody titer level can be based on, for example, animmunobinding assay which measures the concentration of antibodies inthe serum which bind to a specific antigen, for example, LPS and/or LOS.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site (for example, the lungs or systemiccirculation). Higher or lower concentrations can be selected based onthe mode of delivery, for example, trans-epidermal, rectal, oral,pulmonary, or intranasal delivery versus intravenous or subcutaneousdelivery. Dosage can also be adjusted based on the release rate of theadministered formulation, for example, of an intrapulmonary spray versuspowder, sustained release oral versus injected particulate ortransdermal delivery formulations, and so forth. To achieve the sameserum concentration level, for example, slow-release particles with arelease rate of 5 nanomolar (under standard conditions) would beadministered at about twice the dosage of particles with a release rateof 10 nanomolar.

The methods of using conjugates, and the related compositions andmethods of the disclosure, are useful in increasing resistance to,preventing, ameliorating, and/or treating infection and disease causedby S. sonnei in animal hosts, and other, in vitro applications. Theseimmunogenic compositions can be used for active immunization forprevention of infection, and for preparation of immune antibodies. Theimmunogenic compositions are composed of non-toxic components, suitablefor infants, children of all ages, and adults.

The methods of the disclosure are broadly effective for treatment andprevention of bacterial disease and associated inflammatory, autoimmune,toxic (including shock), and chronic and/or lethal sequelae associatedwith bacterial infection. Therapeutic compositions and methods of thedisclosure for prevention or treatment of toxic or lethal effects ofbacterial infection are applicable to a wide spectrum of infectiousagents. Non-lethal toxicities that will be ameliorated by these methodsand compositions can include fatigue syndromes, inflammatory/autoimmunesyndromes, hypoadrenal syndromes, weakness, cognitive symptoms andmemory loss, mood symptoms, neurological and pain syndromes andendocrine symptoms. Any significant reduction or preventive effect ofthe conjugate with respect to the foregoing disease condition(s) orsymptom(s) administered constitutes a desirable, effective property ofthe subject composition/method of the disclosure.

The instant disclosure also includes kits, packages and multi-containerunits containing the herein described pharmaceutical compositions,active ingredients, and/or means for administering the same for use inthe prevention and treatment of bacterial diseases and other conditionsin mammalian subjects. Kits for diagnostic use are also provided. In oneembodiment, these kits include a container or formulation that containsone or more of the conjugates described herein. In one example, thiscomponent is formulated in a pharmaceutical preparation for delivery toa subject. The conjugate is optionally contained in a bulk dispensingcontainer or unit or multi-unit dosage form. Optional dispensing meanscan be provided, for example a pulmonary or intranasal spray applicator.Packaging materials optionally include a label or instruction indicatingfor what treatment purposes and/or in what manner the pharmaceuticalagent packaged therewith can be used.

The subject matter of the present disclosure is further illustrated bythe following non-limiting Examples.

EXAMPLES Example 1

Growth of Bacteria and Isolation of LPS.

S. sonnei strain 53G and P. shigelloides strain 7-63 (serotype 017) wereobtained from Sam Formal (Walter Reed Army Institute of Research, SilverSpring, Md.) and cultivated as described by Taylor, et al. (Infect Immun61:3678-3687, 1993). LPS from either strain was extracted by the hotphenol method and purified as described by Westphal and Himmelspach(1983, Prog Allergy 33:9-39, incorporated herein by reference in itsentirety).

Isolation of Oligosaccharides.

S. sonnei LPS (200 mg) was treated with 1% acetic acid at 100° C. for1.5 hours. Lipid A was removed by ultracentrifugation at 142,000×g for 5hours at 4° C., and the soluble product subjected to gel chromatographyon a BIOGEL® P-10 (1×100 cm) column in pyridine/acetic acid/water buffer(4:8:988 mL), monitored with a Knauer differential refractometer.

Analytical Methods.

Protein concentration was measured by the method of Lowry et al. (J BiolChem 193:265-275, 1951), and sugar concentration was measured by theanthrone assay (Scott and Melvin, Anal Chem 25:1656-1661, 1953).SDS/PAGE used 14% gels according to the manufacturer's instructions(BIO-RAD®). Immunodiffusion was performed in 1% agarose in phosphatebuffered saline (PBS).

Spectroscopy.

¹H and ¹³C NMR spectra were recorded by using a Varian Inova 500-MHzspectrometer for samples in D₂O solutions at 35° C. with acetonestandard (2.225 ppm for ¹H and 31.5 ppm for ¹³C) using standard pulsesequence COSY, TOCSY (mixing time 120 ms), NOESY (mixing time 200 ms),and Heteronuclear Single Quantum Coherence and Heteronuclear MultipleBond Correlation (100-ms long range transfer delay). Capillaryelectrophoresis (CE)-MS was obtained on a 4000 QTRAP mass spectrometer(Applied Biosystems/MDS Sciex) with a Prince CE system (PrinceTechnologies) with a 90-cm length bare fused-silica capillary using 15mM ammonium acetate in deionized water, pH 9.0, as an injection module.A sheath solution (isopropanol/methanol, 2:1) was delivered at a flowrate of 1.0 μL/min. A 5-kV electrospray ionization voltage was used fornegative ion detection modes. For some experiments samples weredephosphorylated at 1 mg/mL of 40% hydrofluoric acid (HF) for 24 hoursat 4° C. MALDI-TOF mass spectra of the derivatized proteins and theconjugates were obtained with an OmniFlex MALDI-TOF instrument (BrukerDaltonics) operated in the linear mode. Samples for analysis weredesalted, and 1 μL was mixed with 20 μL of sinnapinic acid matrix madein 30% CH₃CN and 0.1% trifluoroacetic acid. Next, 1 μL of mixture wasdried on the sample stage and placed in the mass spectrometer.

Preparation of Conjugates.

To 15 mg of Bovine Serum Albumin (BSA) (Sigma) or recombinant DiphtheriaToxin (rDT) [CRM H21G (Schneerson et al., J Exp Med 152:361-376, 1980)]in 2.2 mL of buffer A (PBS, 0.1% glycerol, 5 mMethylendiaminetetraacetic acid (EDTA), pH7.2), 4 mg of N-succinimidyl3-(bromoacetamido)propionate (SBAP; Pierce) in 40 μL of DMSO was addedand reacted at pH 7.2 in room temperature with mixing for 1.5 hours.Next, the solution was applied to a SEPHADEX™ G-50 column (1×50 cm) inPBS, and the void volume fraction (Pr—Br) was concentrated by using anAmicon Ultra-15 centrifuge filter device (MILLIPORE®) to 2.6 mL (13 mgrecovered), and 0.1 mL was removed for analysis. To 12 mg of Pr—Br in2.4 mL of buffer A, 10 mg of O-(3-thiopropyl)hydroxylamine was added in300 μL of 1 M KCl and reacted at pH 7.2 in room temperature with mixingfor 3 hours. Next, the solution was passed through the SEPHADEX® G-50,and the void volume fraction (Pr—ONH₂) was concentrated to 2.6 mL asabove, and 0.2 mL was removed for analysis. Ten milligrams of Pr—ONH₂was reacted with 25 mg (7.8 μmol) O-SPC in 3 mL of buffer A overnight,at pH 7.2, in room temperature with mixing. The solution was then passedthrough the SEPHAROSE® G-75 (1×100 cm) in PBS, and the void volumefraction was collected and analyzed for sugar and protein contents andmolecular mass by MALDI-TOF and SDS/PAGE. Three conjugates were obtainedthis way: 1, BSA/O-SPC-F2; 2, rDT/O-SPC-F2; and 4, BSA/O-SPC-F3. Forpreparation of conjugate 3, binding of rDT-ONH₂ and O-SPC-F2 was done in1.5 mL of buffer A, and the product (rDT/O-SPC-F2) contained twice thenumber of O-SPC-F2 chains per rDT molecule as conjugate 2 (12 vs. 6).Conjugates of full-length O-SP bound by multiple attachments to eitherrecombinant Bacillus anthracis protective antigen or rEPA were preparedas described by Passwell, et al., Pediatr Inf Dis J22:701-706, 2003).

Immunization.

Five- to 6-week-old female National Institutes of Health Swiss—Webstermice were injected subcutaneously (s.c.) 3 times at 2-week intervalswith 2.5 μg of saccharide as a conjugate in 0.1 mL of PBS. Groups of 10mice were exsanguinated 7 days after the second or third injections asdescribed by Schneerson et al. J Exp Med 152:361-376, 1980. Controlsreceived PBS.

Antibodies.

Serum IgG antibodies were measured by ELISA using S. sonnei or P.shigelloides LPS as a coating antigen as described by Plikaytis andCarlone, 2005, Program ELISA for Windows User's Manual (Centers forDisease Control, Atlanta), Version 2). The results were computed with anELISA data processing program provided by the Biostatistics andInformation Management Branch, Centers of Disease Control, Atlanta(Johnson and Nicholls, 1994, J Biol Chem 269:4349-4354). A polyclonalrabbit antiserum obtained by immunizing rabbits with multipleintravenous (i.v.) injections of heat-killed S. sonnei bacteria was usedin the immunodiffusion assays.

Competitive Inhibition ELISA.

Inhibition was assayed by incubating conjugate induced mice sera,diluted to the concentration that gave an absorbance of μ 1.0 at awavelength of 405 nm (A405), with 2, 10, or 50 μg of inhibitor (Table 2)per well, for 1 hour at 37° C. and overnight at 4° C. The ELISA was thencontinued as usual. Sera at the same dilution with and without inhibitorwere compared. Percentage inhibition was defined as (A405 adsorbedserum/A405 nonadsorbed serum) X 100%. Core-free P. shigelloides O-SP wasobtain by treating the O-SP with anhydrous HF for 1 hour at 25° C.Haemophilus influenzae type a capsular polysaccharide was used as acontrol.

Statistics.

GMvalues of the groups of 10 mice were calculated. Unpaired t test wasused to compare GMs between different groups of mice.

Isolation and Chemical Characterization of O-SPC.

LPS was extracted from 18-hour cultures of S. sonnei or Plesiomonasshigelloides as described by Taylor, et al., (Infect Immun 61:3678-3687,1993). S. sonnei saccharides, released after mild acid hydrolysis fromlipid A, were separated into 4 fractions (FIG. 1). The yields offractions 1-4 were 50%, 17%, 31%, and 2% by weight, respectively.Integration of the FucNAc4N methyl signal in 1H-NMR spectra (1.34-1.36ppm) relative to the anomeric signals of core α-Gal M (5.82 ppm) andα-Gal L (5.62 ppm) (see Table 1 and FIG. 4 Scheme 1) showed thatfraction F1 contained core with ≈29 O-SP repeat units (RU), F2 containedcore with an average of 3.5 RU, and F3 contained core with an average of1.3 RU (FIG. 2). Fraction F4 contained various degradation products andwas not studied further.

MS spectra confirmed that fraction F3 consisted of the core with 1 RU(FIG. 3A). The following species have been detected: core+1 RU withoutthe core GlcN (2,110.6 Da), core+1 RU including GlcN (2,271.7 Da),core+1 RU including GlcN but without phosphate (P) (2,190.9 Da), core+1RU with GlcN, P and PEtN (2,377.8 Da), and the latter but without P(2,314.1 Da).

MS analysis of fraction F2 showed a complex spectrum containing 3×, 4×,and 5× charged ions of the oligosaccharides with 2, 3, and 4 RU,heterogenic in P, PEtN, and GlcN substitutions, respectively. Tointerpret the spectrum, F2 was partially dephosphorylated. The followingspecies were detected (FIG. 3B): (i) core+2 RU (2,433.9 Da), andpartially substituted with P (2,496.9 Da), and core+2 RU with GlcN(2,595.0 Da); (ii) core+3 RU (2,837.1 Da), and partially substitutedwith a phosphate group (2,918.1 Da), or core+2 RU with GlcN (2,999.1Da); and (iii) core+4 RU (3,240.6 Da). Integration of NMR data indicatedthat even longer oligosaccharides, with 5 RU were present in F2 buttheir signals were not observed in the mass spectra, probably becausethey showed multiply-charged ions mixed with background signals. The F2and F3 oligosaccharide fractions were used for conjugation.

Preparation and Characterization of Conjugates.

Three conjugates were prepared by binding O-SPC-F2 to either BSA(conjugate 1; BSA/O-SPC-F2) or recombinant diphtheria (rDT) toxin(conjugates 2 and 3: rDT/O-SPC-F2). One conjugate was prepared bybinding O-SPC-F3 to BSA (conjugate 4: BSA/OSPC-F3).

The conjugation was based on formation of stable oxime linkages betweenthe Kdo residue present at the O-SPC reducing end and an aminooxy linkerbound to the carrier protein. This procedure yielded high molecular massconjugates, revealed by MALDI-TOF MS, SDS/PAGE, and protein and sugarcolorimetric assays; all methods provided comparable results. The numberof O-SPC chains per protein was calculated from the molecular mass ofthe conjugate, the carrier protein, and the O-SPC. Thus, conjugate 1contained +7 O-SPC chains/BSA molecule, conjugate 2 contained +6O-SPC/rDT, conjugate 3 contained +12 O-SPC chains/rDT, and conjugate 4contained +11 O-SPC chains/BSA (Table 2). An excess of saccharide wasused for conjugation to ensure maximal binding. The yield of the proteinin the conjugate was 65-75%, and the yield of the saccharide was 30-35%.Conjugates 1, 2, and 3 prepared with O-SPC-F2 reacted by doubleimmunodiffusion with rabbit anti-S. sonnei and anti-protein sera by aline of identity. Conjugate 4 prepared with O-SPC-F3 precipitated withthe anti-BSA serum but not with the anti-S. sonnei serum. Onlyconjugates of O-SPC-F2 were used for immunization.

IgG Anti-LPS Responses (Table 2).

Conjugates 1, 2, and 3 elicited low levels of IgG anti-LPS after thesecond injection with a booster response after the third. The geometricmeans (GM) of IgG anti-LPS after the third injection were 366 ELISAunits (EU) for conjugate 1 and 392 EU for conjugate 2. Conjugate 3,which contained twice as much of O-SPC-F2 chains per rDT than conjugate2 (12 vs. 6), induced statistically-lower GM antibody levels (150 EU vs.392 EU; P<0.01). All 3 O-SPC-F2 conjugates induced statistically-higherantibody levels than the “lattice”-type conjugate (a clinical lot)prepared with the full-length O-SP (366 vs. 67, P<0.0001; 392 vs. 67,P<0.0001; 150 vs. 67: P<0.05).

IgG Anti-O-SP Responses (Table 3).

Coating the ELISA plates with S. sonnei or P. shigelloides LPS ofidentical O-SP but different sera induced by either O-SPC or O-SPconjugates were inhibited similarly by O-SPs of S. sonnei and of P.shigelloides with or without the core.

IgG Anti-O-SP Responses (Table 4).

Coating the ELISA plates with S. sonnei or P. shigelloides LPS ofidentical O-SP but different sera induced by either O-SPC or O-SPconjugates were inhibited similarly by O-SPs of S. sonnei and of P.shigelloides with or without the core.

Example 3 Treatment of Subjects with S. sonnei O-PS Conjugate

This example describes methods that can be used to treat a subject thathas or is at risk of having an infection from S. sonnei byadministration of one or more of the disclosed conjugates. In particularexamples, the method includes screening a subject having, thought tohave, or at risk of having (for example due to impaired immunity,physiological status, or exposure to S. sonnei) a S. sonnei infection.Subjects of an unknown infection status can be examined to determine ifthey have an infection, for example using serological tests, physicalexamination, enzyme-linked immunosorbent assay (ELISA), radiologicalscreening or other diagnostic technique known to those of skill in theart. In some examples, a subject is selected that has a S. sonneiinfection or is at risk of acquiring a S. sonnei infection. Subjectsfound to (or known to) have a S. sonnei infection and thereby treatableby administration of the disclosed conjugates are selected to receivethe conjugates peptide. Subjects may also be selected who are at risk ofdeveloping a S. sonnei infection for example, the elderly, theimmunocompromised and the very young, such as infants.

Subjects selected for treatment can be administered a therapeutic amountof disclosed conjugate. The conjugate can be administered at doses of 1μg/kg body weight to about 1 mg/kg body weight per dose, such as 1 μg/kgbody weight-100 μg/kg body weight per dose, 100 μg/kg body weight-500μg/kg body weight per dose, or 500 μg/kg body weight-1000 μg/kg bodyweight per dose or even greater. However, the particular dose can bedetermined by a skilled clinician. The agent can be administered inseveral doses, for example continuously, daily, weekly, or monthly.

The mode of administration can be any used in the art. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. An immunogenic conjugate comprising the structure of:Pr—Sr—O—N═C-Kdo-OS wherein Pr is a carrier protein, Sr is an optionalspacer moiety, Kdo is 3-deoxy-D-manno-octulosonic acid, and OS comprisesat least one oligosaccharide or polysaccharide obtained from S. sonnei.2. The immunogenic conjugate of claim 1, wherein the oligosaccharide orpolysaccharide includes a core oligosaccharide.
 3. The immunogenicconjugate of claim 2, wherein the oligosaccharide core, comprises:

wherein R is between 1 and 10 disaccharide repeat units.
 4. Theimmunogenic conjugate of claim 3, wherein the oligosaccharide core,consists of:

wherein R is between 1 and 10 disaccharide repeat units.
 5. Theimmunogenic conjugate of claim 3, wherein the disaccharide repeat unitcomprises:α-L-AltNAcA-3-β-FucNAc4N-4-.
 6. The immunogenic conjugate of claim 5,wherein the disaccharide repeat unit consists of:α-L-AltNAcA-3-β-FucNAc4N-4-.
 7. The immunogenic conjugate of claim 5,wherein the conjugate consists of 1-10 disaccharide repeat units.
 8. Theimmunogenic conjugate of claim 5, wherein the conjugate comprises 3-5disaccharide repeat units.
 9. The immunogenic conjugate of claim 1,wherein the conjugate comprises between 1 and 20 oligosaccharides percarrier protein molecule.
 10. The immunogenic conjugate of claim 9,wherein the conjugate comprises between 1 and 14 oligosaccharides percarrier protein molecule.
 11. The immunogenic conjugate of claim 1,wherein the protein carrier comprises recombinant diphtheria toxin(rDT).
 12. The immunogenic conjugate of claim 1, wherein the proteincarrier comprises recombinant Pseudomonas aeruginosa Exoprotein A(rEPA).
 13. The immunogenic conjugate of claim 1, wherein the proteincarrier comprises recombinant C. difficile toxin B repeat (rBRU).
 14. Atherapeutic composition comprising the conjugate of claim 1 and apharmaceutically acceptable carrier.
 15. A method of eliciting an immuneresponse in a subject, comprising administering to the subject theimmunogenic conjugate of claim
 1. 16. A method of treating and/orinhibiting an infection by S. sonnei in a subject, comprising:administering to the subject the immunogenic conjugate of claim
 1. 17. Atherapeutic composition comprising the conjugate of claim 8 and apharmaceutically acceptable carrier.
 18. A method of treating and/orinhibiting an infection by S. sonnei in a subject, comprising:administering to the subject the immunogenic conjugate of claim
 8. 19.The immunogenic conjugate of claim 8, wherein the conjugate comprisesbetween 1 and 14 oligosaccharides per carrier protein molecule.